Primary study on the effect of glucose on mouse CD4+ T cell differentiation and its mechanism / 上海交通大学学报（医学版）

Objective :To study the effect of glucose on mouse CD4+ T cell differentiation. Methods :Mouse naïve CD4+ T cells cultured in the regulatory T cell (Treg), Th1, Th17 or Th2 differention condition were treated with different concentrations of glucose for 5 days. Treg, Th1, Th17 or Th2 percentages were measured by flow cytometry. Quantitative real-time PCR was used to detect the gene expressions of related cytokines and transcriptional factors. Results :The proportions of Treg and Th2 as well as the gene expressions of transforming growth factor-β, interleukin-4 (IL-4) and IL-13, and transcriptional factors, Foxp3 (forkhead box P3) and Gata3 (GATA binding protein 3), were increased significantly with the treatment of increasing concentration of glucose. On the contrary, with the glucose treatment, the percentages of Th1 and Th17 were reduced, and the gene expressions of the related cytokines and cytokine receptors, such as interferon-γ, IL-17A, IL-17F, IL-22 and IL-23R, and the related transcriptional factors, Tbx21 (T-box transcription factor 21) and RORC (RAR related orphan receptor C), were decreased consistently. Conclusion :Glucose promotes Treg and Th2 differentiation while inhibits Th1 and Th17 differentiation in vitro.

Current Status of Stem Cell Treatment for Type I Diabetes Mellitus

BACKGROUND: Diabetes mellitus is a major health concern in current scenario which has been found to affect people of almost all ages. The disease has huge impact on global health; therefore, alternate methods apart from insulin injection are being explored to cure diabetes. Therefore, this review mainly focuses on the current status and therapeutic potential of stem cells mainly mesenchymal stem cells (MSCs) for Type 1 diabetes mellitus in preclinical animal models as well as humans. METHODS: Current treatment for Type 1 diabetes mellitus mainly includes use of insulin which has its own limitations and also the underlying mechanism of diseases is still not explored. Therefore, alternate methods to cure diabetes are being explored. Stem cells are being investigated as an alternative therapy for treatment of various diseases including diabetes. Few preclinical studies have also been conducted using undifferentiated MSCs as well as in vitro MSCs differentiated into β islet cells. RESULTS: These stem cell transplant studies have highlighted the benefits of MSCs, which have shown promising results. Few human trials using stem cells have also affirmed the potential of these cells in alleviating the symptoms. CONCLUSION: Stem cell transplantation may prove to be a safe and effective treatment for patients with Type 1 diabetes mellitus.

Optimization of in vitro human follicular helper T cell differentiation condition / 中国药科大学学报

In order to explore the regulation mechanisms of follicular helper T cell (Tfh Cell) differentiation,optimized conditions of in vitro induction from both peripheral blood mononuclear cells and MAC sorted Na(i)ve CD4 + T cells to human Tfh cells were developed.Induction efficiency difference of TCR signal anti-hCD3e stimulation between coated on solid phase and in soluble phase was also determined.Differentiation efficiency of CD4 + CXCR5 + ICOS+PD-1 + Tfh cell was determined by FACS while the expression level of IL-21 in cell supernatant was determined by ELISA tests.An ultimate induction condition that 5 μg/mL coated overnight anti-hCD3e stimulated na(i)ve CD4 + T cells to differentiate into Tfh at an up to 20.4％ percentage was finally determined.The optimization of in vitro induction protocol of human Tfh provided an effective examine platform for the studies on Tfh differentiation mechanisms and related pharmacology,toxicity and metabolic experiments.

The efficiency and function detection of NK cell differentiation from human umbilical cord hematopoietic stem cells in vitro / 天津医药

Objective To detect the efficiency and function of NK cell differentiation from human umbilical cord he?matopoietic stem cells (HSCs) in vitro. Methods CD34+hematopoietic stem cells were isolated from human umbilical cord blood, and inoculated into SCGM medium containing 20 μg/L FMS like tyrosine kinase 3 ligand (Flt-3L), stem cell fac?tor (SCF), interleukin (IL)-7, IL-15 and IL-21. And CD34+HSCs were differentiated into NK cells in directional inducing. The growth state of cells was observed. The expressions of CD56, NKG2D, NKp46, CD3, CD19 and CD34 were detected by flow cytometry in the differentiation of 7, 14, 21 and 28 d. In the differentiation of 21 d and 28 d, the differentiation cells were used as effector cells, and K562 cells as target cells. The ratios of effector cells and target cells were 8∶1, 4∶1, 2∶1 and 1∶1. The killing activity of the differentiated cells was detected by lactate dehydrogenase (LDH) cell toxicity assay and 7AAD/CFSE labeling method. Results CD34+HSCs derived from human umbilical cord blood can proliferate in vitro under appropriate condition. There were no significant differences in the expression of CD3 and CD19 between different differentia?tion stages (7, 14, 21 and 28 d, P>0.05). The expressions of CD56, NKG2D and NKp46 were significantly different (P<0.05), and the ultimate expression amount was (72.57±1.60)%, (32.83±1.29)%and (29.53±2.40)%. The expression of CD34 decreased gradually, and the lowest was (12.13 ± 2.01)%. The maximum killing activity detected by LDH cell toxicity assay and 7AAD/CFSE labeling method reached(49.91±2.76)%and (40.87±1.12)%.The killing activity of NK cells was decreased in the order of 8∶1, 4∶1, 2∶1 and 1∶1 groups (P<0.05). There was no significant difference in the killing activity between NK cells of 28 d and 21 d. Conclusion Human umbilical cord hematopoietic stem cells can differentiate into NK cells un? der appropriate conditions in vitro, and the NK cells induced from differentiation are with killing activity.

Comparative Analysis of Dibutyric cAMP and Butyric Acid on the Differentiation of Human Eosinophilic Leukemia EoL-1 Cells

Purification of enough numbers of circulating eosinophils is difficult because eosinophils account for less than 5% peripheral blood leukocytes. Human eosinophilic leukemia EoL-1 cells have been considered an in vitro source of eosinophils as they can differentiate into mature eosinophil-like cells when incubated with dibutyryl cAMP (dbcAMP) or butyric acid. In this study, the viability and phenotypic maturation of EoL-1 cells stimulated by either dbcAMP or butyric acid were comparatively analyzed. After treatment with 100 microM dbcAMP or 0.5 microM butyric acid, EoL-1 cells showed morphological signs of differentiation, although the number of nonviable EoL-1 cells was significantly increased following butyric acid treatment. Stimulation of EoL-1 cells with 0.5 microM butyric acid more effectively induced the expression of mature eosinophil markers than stimulation with dbcAMP. These results suggest that treatment of EoL-1 cells with 0.5 microM butyric acid for limited duration could be an effective strategy for inducing their differentiation. Considering that expression of CCR3 was not sufficient in EoL-1 cells stimulated with 0.5 microM butyric acid, treatment of the chemically stimulated EoL-1 cells with cytokines, which primarily support eosinophil maturation, would help to obtain differentiated EoL-1 cells with greater functional maturity.

Transcriptional Profiles of Imprinted Genes in Human Embryonic Stem Cells During In vitro Differentiation

BACKGROUND AND OBJECTIVES: Genomic imprinting is an inheritance phenomenon by which a subset of genes are expressed from one allele of two homologous chromosomes in a parent of origin-specific manner. Even though fine-tuned regulation of genomic imprinting process is essential for normal development, no other means are available to study genomic imprinting in human during embryonic development. In relation with this bottleneck, differentiation of human embryonic stem cells (hESCs) into specialized lineages may be considered as an alternative to mimic human development. METHODS AND RESULTS: In this study, hESCs were differentiated into three lineage cell types to analyze temporal and spatial expression of imprinted genes. Of 19 imprinted genes examined, 15 imprinted genes showed similar transcriptional level among two hESC lines and two human induced pluripotent stem cell (hiPSC) lines. Expressional patterns of most imprinted genes were varied in progenitors and fully differentiated cells which were derived from hESCs. Also, no consistence was observed in the expression pattern of imprinted genes within an imprinting domain during in vitro differentiation of hESCs into three lineage cell types. CONCLUSIONS: Transcriptional expression of imprinted genes is regulated in a cell type-specific manner in hESCs during in vitro differentiation.

In vitro differentiation of embryonic stem cells into cardiac-like and neuronal-like cells / Indução de diferenciação in vitro de células-tronco embrionárias em células de tecido cardíaco e nervoso

Embryonic stem cells are pluripotent cell lines with the capacity of self-renewal and a broad differentiation plasticity. They are isolated from preimplantation embryos and can be cultured in vitro for long time without losing their pluripotency. Embryonic stem cells can also differentiate in vitro with the proper combination of growth and differentiation factors, cells will differentiate into more advanced stages of embryogenesis generating different adult cell type. In the present study, we induced the in vitro differentiation of mouse embryonic stem cells (line R1) into cardiomyocytes and neuronal cells. These differentiations were evaluated by reverse transcription-polymerase chain reaction to verify presence of tissue-specific markers.

Células-tronco embrionárias são linhagens celulares pluripotentes capazes de se multiplicar indefinidamente e com grande capacidade de diferenciação celular. São isoladas de embriões em estágio pré-implantacional e podem ser cultivadas por longo tempo em laboratório sem perder sua pluripotencialidade. Células-tronco embrionárias podem, ainda, se diferenciar in vitro através da adição de fatores de crescimento e diferenciação ao meio de cultivo. As células se diferenciarão em estágios mais avançados de embriogênese, gerando tipos diferentes de células adultas. No presente estudo, induzimos a diferenciação in vitro de células-tronco embrionárias de camundongos (linhagem R1) em células de tecido cardíaco e nervoso. A diferenciação foi avaliada pela reação em cadeia da polimerase precedida de transcrição reversa para verificar a presença de marcadores tecido-específicos.

Derivation, culture and in vitro differentiation of human embryonic stem cells / 国际生物医学工程杂志

Human embryonic stem(hES) cells can self-renew and have the ability to differentiate into any type of cells of the body. These characteristics make hES cells a good candidate for cell-based therapies. Current techniques for derivating and culturing embryonic stem cells are very mature. However, concerns arise that pathogen contamination may make these cells unsuitable for therapeutic purposes. An optimal growth environment is greatly needed. Through various in vitro differentiation methods, human embryonic stem cells can be induced into many specialized cell types. However the mechanism of committed differentiation is still unknown.